Our laboratory has discovered that Na+ binding is required for the procoagulant, but not the anticoagulant activity of thrombin, and has elucidated the structural basis of this effect. This discovery has in turn enabled the rational engineering of thrombin mutants selectively compromised in their procoagulant properties. Recently, one of these mutants was tested in a primate model of platelet-dependent thrombosis and has shown safe and potent anticoagulant and antithrombotic effects, consistent with the predictions from in vitro studies. We are now in a position to bring the basic knowledge garnered from our in vitro studies and the exciting new developments of our preliminary in vivo studies to an entirely new level. In the proposed research project, we plan to rationally engineer new thrombin mutants with exclusive activity toward the anticoagulant protein C. We will thoroughly characterize these mutants in vitro and structurally. We will then use these mutants, together with the one already tested in vivo, to assess their effects on thrombosis and hemostasis in baboons and how in vivo properties correlate to those predicted from in vitro studies. We will also compare the effects of these mutants with the direct administration of activated protein C and other antithrombotic agents. We will finally use these mutants as tools to selectively deplete the plasma protein C pool and to assess the consequences of this depletion on the homeostatic balance between coagulation and anticoagulation. Developments from the proposed research plan will reveal new and rational strategies to re-engineer thrombin specificity and will have an enormous impact on molecular enzymology in general. In addition, they will reveal the true pharmacologic potential of engineered thrombin mutants in the control of clinical disorders that involve coagulation and thrombosis. Finally, these studies will produce valuable reagents to further elucidate the role of protein C in balancing the response of thrombin to vascular injury [unreadable] [unreadable]